The bulk market: for .beta.-lactam antibiotics is a highly competitive field and one of the parameters which influence the price is the purity of the product. Therefore, it is very important for a producer to have access to a production method which gives a high yield of a very pure product.
Presently, most of the cephalosporins used are so-called semi-synthetic products. This designation implies that they are obtained by modifying a .beta.-lactam product obtained by fermentation by one or more chemical reactions. Typically, one of the chemical reactions involved in this modification is acylation of the 7-amino group of an intermediate cephalosporin nucleus the parent of which is originally obtained by fermentation and optionally further modified in other chemical reactions.
In the following, the acyl group introduced at the 7-amino group is referred to as the cephalosporin side chain or just the side chain. The acid corresponding to the side chain is designated the side chain acid. The designation "a cephalosporin nucleus" designates a compound the acylation of which with the cephalosporin side chain results in the formation of a .beta.-lactam antibiotic or, more specifically, a cephalosporin.
The acylation can be performed by reacting an optionally protected form of the cephalosporin nucleus with a derivative--e.g. the acid chloride--of an optionally protected form of the cephalosporin side chain in an organic solvent--typically methylene chloride--in the presence of a base. As an alternative to the acid chloride a mixed anhydride can be used for the acylation. In both cases, the acylation has to be carried out under anhydrous conditions at a temperature which should preferably be below zero.
The acylation reaction never comes to completion and at the point when the work up is started, the reaction mixture will contain the desired product as well as unreacted starting materials and possible byproducts. After the acylation, the protective groups, if any, have to be removed either in situ or after the protected intermediate product has been isolated. The working up of the intermediate product and the removal of the protective groups generally involves at least one step in which the product is contacted with water or an aqueous solvent. During the deprotection operation great care must be taken to avoid cleavage of the acyl bond just established.
The final purification of .beta.-lactam antibiotics can be hampered by the fact that the acid-base properties and the solubility of some of the impurities contained in the crude products are not very much different from the similar properties of the desired product. This implies that co-precipitation of impurities may easily take place when the crude product is reprecipitated or recrystallized and it can therefore be difficult to achieve a high purity of the product and a high yield at the same time.
As an alternative to the methods outlined above, the side chain can be introduced by an enzyme catalyzed acylation. In this case either the free acid corresponding to the side chain or an activated derivative thereof such as the amide or a lower alkyl ester can be utilized as the source of the acyl group. The enzymatic acylation can be carried out at ambient temperature and thus, the expense and the inconvenience connected with working at low temperature is eliminated. The solvent is water or a mixture of water with water-miscible organic solvents. The stability of the desired products and the rate of the acylation reaction both depend on the pH value in the reaction mixture. Thus, at a pH value of about 4, the desired products are fairly stable and the rate of the acylation reaction is fairly low. At a pH value of about 7, deacylation of the desired product progresses and at the same time the rate of the acylation reaction is high. This means that the pH value at which the enzymatic acylation is most favourably carried out under ordinary conditions constitutes a compromise between these oppositely directed preferences.
A method for the purification of cephalexin is described in U.S. Pat. No. 4,003,896 (to NOVO INDUSTRI A/S). According to this patent, naphthalene or a naphthalene derivative is added to an aqueous solution containing crude cephalexin to provide a sparingly soluble complex of naphthalene or of the naphthalene derivative with cephalexin. This complex is isolated and decomposed to provide a high yield of very pure cephalexin. However excellent the purification method described works, it can not but secure a good yield of the purification step.